2) Project Summary/Abstract The precise organization of cells in the nervous system ensures full functionality of the human brain. However, there is a critical gap in our understanding of how diverse glial populations dynamically contribute to brain cellular organization and function. During construction of the brain, and throughout the nervous system, glial and neuronal cells choreograph their maturation to create functional circuits. How this glial and neuronal organization is choreographed to ensure proper circuits are created while improper ones are eliminated is poorly understand. A major obstacle in understanding this basic neuroscience is the fields incomplete understanding of glial cell diversity. Filling this major gap is critical to fully understanding how neural cells assemble to create functional nerves. To address this, we have developed imaging paradigms that will divulge the diversity of glial cells in the nervous system and thus fill in a major gap in basic neuroscience. Unique from other approaches, we have profiled oligodendrocytes using time-lapse imaging of their dynamics. We apply this time-lapse format to two new imaging platforms that we developed in close collaboration with engineers. One of these platforms is a super-resolution techniques that will reveal distinct interactions that individual glial populations have throughout their maturation. Importantly, this new super-resolution techniques allow us to visualize glial cells in their native physiological state. The second platform provides unique insight into the physiological properties of individual cells or compartment of cells, throughout their maturation process. We combine those imaging approaches with genetic screens, circuit analysis and behavioral tests. Combined with these technical advances, conceptually we provide innovation by considering the role of glial cells beyond their traditional textbook definitions. As a proof of principle that this approach will provide new cutting edge information about the brain, we used it to identify an undescribed oligodendrocyte population that interacts with sensory neurons. Our data show that during construction of the nervous system, these cells experience distinct interactions with sensory neurons that ultimately guides their maturation; a process we can disrupt by preventing the glial cells from contacting sensory neurons. The approaches presented in this proposal are important to the field as they converge molecular, cellular, anatomical and behavioral components to understand glial diversity and its role in brain function. The central hypothesis in these discoveries is that glial cells initiate precise, diverse and dynamic interactions during development to ensure genesis of the correct neuronal circuits that drive precise behaviors. The rationale of this proposal is that our proposed experiments will define the dynamic mechanisms that control nervous system assembly in an intact vertebrate. The long-term goal is to leverage these studies in the nervous system to understand how the brain works while also improving diagnosis and therapeutics in the clinic. We will introduce new theories of nerve assembly by addressing the following goals: define new glial populations, reveal new physiological properties of glia, identify genes that are essential for glial function, reveal role of these glia in circuits and behavior. The proposed aims are significant because they are expected to expand an understanding of how the precise and dynamic organization of neural cells ensures functionality of the human body.